Methods and Systems for In Situ Calibration of Imaging in Biological Analysis

ABSTRACT

Software, methods, and systems for calibrating photometric devices are provided. These involve using a non-uniform test illumination field to approximate a photon transfer curve by calculating stable pixel values and statistical dispersions on a pixel-by-pixel basis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of patent application Ser. No.10/911,887 filed Aug. 5, 2004, now U.S. Pat. No. 7,045,756, which isincorporated herein by reference.

INTRODUCTION

The present teachings generally relate to methods, software, andapparatus useful in evaluation of photometric sensors, for example inin-situ evaluation of imaging instrumentation in biological instruments.

During imaging operations such as biological analysis involvingnucleotide sequencing or microarray processing, photometric sensors areused to detect signals arising from labeled samples or probe featuresresponsive to selected target analytes. These signals can take the formof electromagnetic emissions that are desirably analyzed to quantifysignal intensities arising from labeled samples or probe features andare subsequently resolved to quantitatively or qualitatively evaluatethe presence of a target analyte within a sample. Frequently, imagesassociated with such biological analyses vary in intensity and are of avery high-resolution to accommodate reading of very detailed images suchas high-density microarrays. Therefore, highly precise imaging isrequired, and such imaging is frequently performed in connection with acharge coupled device (CCD).

The quality of data produced by a CCD-based instrument is tightlycoupled to a particular CCD's performance. Algorithms for data analysis,for example, commonly assume the CCD detector provides a linearresponse. Published limits of detection for a particular instrument relyon assumed CCD noise levels. Characteristics such as CCD noise levelsare tested and confirmed during instrument development, and verifiedduring instrument manufacturing. Regardless of initial testing andverification, however, an instrument's performance in-the-field issusceptible to drift and/or changes in noise levels of a CCD containedwithin the instrument.

Quantitative CCD performance measurement can be performed by measuring aparticular CCD's photon transfer curve. A photon transfer curve involvesa plot of detected signal level versus observed noise in the signallevel. Conventionally, the relationship between signal and noise forgenerating a photon transfer curve has been measured as follows. First,a CCD removed from and external to its corresponding instrument and/oroptical assembly is attached to a special test fixture designed touniformly illuminate the CCD array. A series of detections or images aremeasured to determine the average signal noise for a fixed illuminationlevel. For example, multiple images are recorded at a given illuminationlevel. Each unique combination of two images are subtracted from oneanother to produce a series of difference-images. The signal noise isdetermined by computing the average of the difference-image pixelintensity values. The illumination level is determined either byseparate measurement of the illumination source using a calibrated lightdetector, or by averaging the signal from the CCD detector itself. Eachset of images produces a single point on the photon transfer curve, andthe measurement is repeated over a series of illumination levels and/orexposure times. The photon transfer curve for the CCD is constructed byplotting the observed signal level versus the noise in the signal level.In the shot-noise-limited region of the detector, the noise will scalewith the square root of the signal level, appearing linear when plottedon a log scale. The photon transfer curve enables the measurement of aseries of CCD performance characteristics including read noise, fullwell capacity, and gain.

The traditional method of measuring photon transfer curve requires thatthe CCD detector to be exposed to a fairly uniform illumination field.As the uniformity of the illumination field decreases, known proceduresbecome unworkable. In moderately non-uniform illumination fields (ofapproximately +/−10%), known processes produce a noisy photon transfercurve. In a highly non-uniform illumination field of >+/−20%, it becomesessentially impossible to approximate a photon transfer curve usingknown processes. Moreover, in situ, within a particular instrument, itis not typically possible to produce a uniform illumination fieldsufficient to enable measurement of the photon transfer curve usingknown procedures.

Facing governmental regulations regarding instrument validation, newinstrumentation technologies and methods are required to verifybiological instrument performance. Moreover, components of instrumentsmust be validated as a part of the instrument as a whole. However,field-verification of a CCD or other photometric detector is complicatedby the fact that the photometric detector is contained within a largerinstrument system.

Accordingly, methods, software and apparatus to directly andquantitatively verify photometric detectors in situ are needed toprovide characterization information regarding the particularphotometric detector, allowing instrument failure detection, improvedtroubleshooting, quantitative instrument subsystem characterization, andgenerally improved self-diagnostic capability.

SUMMARY

According to various embodiments, the present teachings involve methods,software, and apparatus for determining operating characteristics forphotometric detectors. In various embodiments, the present teachingsrelate to means for directly verifying the performance of a photometricdetector installed as a subsystem within a larger instrument system.

According to other various embodiments, a method is provided fordetermining operating characteristics of a photometric detector that hasphotosensitive pixels. First, an active surface of the photometricdetector is illuminated with electromagnetic radiation. Next, aplurality of varying brightness readings for the various pixels in theplurality of photosensitive pixels is recorded. Then signal statisticaldispersion values are calculated for the plurality of photosensitivepixels independently on a pixel-by-pixel basis over the plurality ofvarying brightness readings. Next stable intensity values for theplurality of photosensitive pixels are calculated independently on apixel-by-pixel basis over the plurality of varying brightness readings.Further, a photon transfer curve is approximated based on the signalstatistical dispersion values and the stable intensity values.

According to other various embodiments, a method for determiningoperating characteristics of a photometric detector, in situ, within animaging instrument is provided. Such methods optionally further includethe step of providing a proper operation indication regarding thephotometric detector based on the photon transfer curve to indicatewhether the photometric detector is operating properly.

According to yet other various embodiments, apparatus is provided fordetermining operating characteristics of a photometric sensing devicecontained within an instrument. The apparatus comprises a photometricsensor comprising a plurality of photosites capable of sensing intensityof electromagnetic energy at the plurality of photosites. The apparatusalso comprises a test illumination field generator for generating a testillumination field optically coupled to the photometric sensor, the testillumination field comprising regions presenting a range of intensitiesto the photometric sensor. The apparatus also comprises a photometricsensor interface circuit coupled to the photometric sensor, thephotometric sensor interface circuit operable to read out sets ofphotosite intensity values from the plurality of photosites, the sets ofphotosite intensity values corresponding to the intensity ofelectromagnetic energy sensed at the plurality of photosites. And theapparatus also comprises a memory coupled to the photometric sensorinterface circuit, the memory comprising machine readable instructionscomprising read instructions for reading a plurality of varyingbrightness readings of the sets of photosite intensity values whileexposed to the test illumination field; noise determination instructionsfor calculating statistical dispersions of the sets of photositeintensity values corresponding to the plurality of varying brightnessreadings; stable intensity determination instructions for calculatingstable intensity values corresponding to the sets of photosite intensityvalues at the plurality of photosites for the plurality of varyingbrightness readings, and photon transfer curve approximationinstructions for approximating a photon transfer curve corresponding tothe photometric sensor based on the statistical dispersions and thestable intensity values.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 illustrates portions of an exemplary photometric detection systemincluding a photometric sensor for detecting photons scattered oremitted from a test image based on electromagnetic radiation in the formof light;

FIG. 2 illustrates portions of an exemplary imaging system including aphotometric sensor detecting electromagnetic radiation emitted from asource of electromagnetic energy;

FIG. 3 illustrates a flow diagram for producing an exemplary photontransfer curve for use in in-situ evaluation of a photometric detector;

FIG. 4 illustrates an exemplary graph of radiant energy as a function ofposition along a one dimensional array of photometric sensor pixels; and

FIG. 5 illustrates an exemplary photon transfer curve.

DESCRIPTION OF VARIOUS EMBODIMENTS

Reference will now be made in detail to some embodiments, examples ofwhich are illustrated in the accompanying drawings. Wherever possible,the same reference numbers are used throughout the drawings to refer tothe same or like parts.

Specific aspects of the present teachings are described below in thecontext of exemplary biological instrument embodiments. However, it isunderstood that the present teachings are not limited in scope to use inbiological instruments but can be used in connection with any type ofphotometric detection instrumentation employing a photometric detector.To clarify several terms used to disclose the present teachings, severaldefinitions are set forth below.

The terms “photometric detector” or “photometric sensor” as used hereinrefer to any device for measuring luminous intensity, intensity ofelectromagnetic radiation, or number or intensity of incident photons.Examples include, but are not limited to, charge coupled device (CCD)arrays, complementary metal oxide semiconductor photodetectors, linescan cameras, and photometric devices used for example in measuringluminous intensity for spectroscopy.

The term “sample photometric intensity readings” as used herein refersto a group of photometric readings of a pixel or group of pixels on apixel-by-pixel basis to obtain a set of pixel intensity readings forparticular pixels. Gathering a number of readings for a particularpixel, given the same illumination of that pixel, provides informationregarding noise at that particular pixel.

The term “statistical dispersion” as used herein refers to a measure ofnumerical diversity within a particular set of numbers. Statisticaldispersion is zero for a set of identical numbers and increases asdiversity among the set of numbers increases. Measurements ofstatistical dispersion include various examples. One measure ofstatistical dispersion is range, which is the difference between thehighest and lowest number values in the set of numbers. Anothermeasurement of statistical dispersion is “standard deviation,” which isthe square root of variance. Various algorithms are known for computingthe standard deviation and variance of a set of numbers.

The term “signal statistical dispersion values” as used herein refers toa measure of numerical diversity of signal intensity values at aparticular pixel.

The terms “stable intensity value” and “stable pixel intensity value” asused herein refer to a value of intensity at a particular pixel absentnoise. In various embodiments, a stable pixel intensity value can beobtained by calculating the average value of the pixel throughout arange of sample photometric intensity readings.

The term “spatially non-uniform in intensity” as used herein in thecontext of an illumination field refers to an illumination field thatvaries in intensity at different points in space, which is to say thatthe illumination field has some relatively brighter regions and somedarker regions.

The term “spatial intensity” as used herein refers to the intensity ofan illumination field at a particular point in space.

The term “temporally non-uniform in intensity” as used herein refers toan illumination field that changes over time for a particular period oftime.

The term “temporally constant” as used herein refers to an illuminationfield that does not change over time for a particular period of time.

FIG. 1 illustrates portions of an exemplary photometric detection system100 including a photometric sensor 108 for detecting photons scatteredor emitted from a test image 104 based on electromagnetic radiation inthe form of light emitted by the light source 112. In variousembodiments, a photon transfer curve corresponding to the photometricsensor 108 is estimated using an arbitrarily non-uniform illuminationfield, which can generally be produced within most instruments. Invarious embodiments, a photometric detector is exposed to an arbitrarilynon-uniform illumination field that can be provided using, for example atest sample that is specially made for validating the instrument. Forexample, in a microarray reader, a test slide can be provided that has arange of brightness regions in ambient light that can be imaged by theinstrument to produce pixel intensity values that can be used toestimate a photon transfer curve for the microarray reader instrument'sCCD. Other examples of biological analysis that involve imaging include,but are not limited to sequence detection, high-throughput screening,and other biological analysis based on imaging of fluorescent markersattached to nucleic acids that are in close proximity and/or of lowlight intensity. In various embodiments, an actual sample that is usedin an instrument in normal operation generally has sufficientlynon-uniform illumination levels to perform the photon transfer curvevalidation operations, thereby eliminating the need for even a testsample.

In various embodiments, the range of illumination levels received at thephotometric detector is preferably highly non-uniform so that thephotometric detector observes a range of illumination from totaldarkness to several times the full-well capacity of the photometricdetector. In various embodiments, where a particular sample lackssufficient variations in signal intensity, a series of measurements arecombined to obtain illumination levels spanning the entire dynamic rangeof the photometric detector.

In various embodiments, each pixel of a photometric detector is used toproduce a unique point on an estimated photon transfer curve. A seriesof images are measured with a non-uniform illumination field. In variousembodiments, twenty images are measured, and twenty readings areobserved for each individual pixel of a CCD detector. Each pixel has anoise value determined by computing the standard deviation of thepixel's twenty readings, and an illumination value determined byaveraging the twenty readings at each pixel. For a CCD detector withdimensions of 240×320 pixels, 76,800 points are generated on the photontransfer curve according to the present teachings. If an adequate rangeof illumination exists, the entire photon transfer curve is producedwith very high resolution after only twenty images. During the samelength of time, other methods produce a single point on the photontransfer curve.

FIG. 2 illustrates portions of an exemplary imaging system 200 includinga photometric sensor 208 detecting electromagnetic radiation emittedfrom a source of electromagnetic energy 212. In various embodiments, thephotometric sensor 208 receives incident photons in the form ofelectromagnetic energy that is scattered off the source 212 or that thesource 212 emits via fluorescence, chemiluminescence, or other means. Invarious embodiments, the photometric sensor 208 is a CCD containedwithin a biological instrument such as a microarray reader. In variousembodiments, biological instruments can include a genetic analyzer witha CCD for detection of capillary electrophoresis separation, or athermal cycler for detection of real-time PCR amplification or end-pointPCR results. The CCD can be tested during manufacturing to ensure thatnoise values associated with the operation of the CCD fall withinacceptable ranges. Moreover, the instrument itself is tested when it ismanufactured to ensure that the integrated imaging system can performimaging operations to an acceptable level of resolution.

Nevertheless, in the field, the instrument may experience adverseenvironmental conditions or portions of the instrument may becomedamaged over time or with use. In various embodiments, such instrumentsemploy aspects of the present teachings to perform self-diagnosticoperations to validate continued proper operation of the imaging system200 within the overall instrument.

FIG. 3 illustrates a flow diagram for producing an exemplary photontransfer curve for use in in situ validation of a photometric detector.First a set of sample intensity values are received (step 310). It isunderstood that the step of receiving sample intensity values can beperformed within the hardware, software and/or firmware of aninstrument, such as a biological instrument. Moreover, it is understoodthat the step of receiving sample intensity values can involve remoteprocessing on a general purpose computer system that is external to andcoupled to the instrument containing the imaging system. Next, N samplesare captured (step 320). It is understood that N can be any number. Invarious embodiments N is chosen to be twenty, and twenty sample imagesare captured to result in twenty sample photometric intensity readingsfor each pixel in the photometric detector. With twenty readings foreach pixel, stable values and statistical dispersions can be calculatedas set forth below.

Next, it is determined whether the illumination field is sufficientlystable to perform the operations and whether there is a sufficient rangeof brightness and darkness in the illumination field (step 330). Invarious embodiments, typical samples that are analyzed in a particularinstrument contain sufficient brightness ranges to measure the photontransfer curve for a particular photometric detector. In variousembodiments, the test for stability of step 330 simply determineswhether the illumination field is sufficiently temporally constant sothat the statistical dispersions measured at particular pixelscorrespond to noise in the photometric detector and shot noise of theradiation source rather than temporal changes in the illumination field.It is understood that some limited temporal changes in the illuminationfield may exist, and that the illumination field can vary with time solong as changes in the illumination field occur at a frequencysubstantially lower than the frequency of sampling used at step 320. Inthis situation, the set of samples measured in step 320 is analyzed toseparate low frequency components produced by drift in the illuminationfield and higher frequency components produced by noise sources.

Next stable intensities and statistical dispersions are calculated forthe detected range of intensities (step 350). In various embodiments,this is performed on a pixel-by-pixel basis by calculating the standarddeviation of intensities measured at each pixel to measure the noise atthe particular pixel. Then the signal value at the pixel is measured byaveraging the value of intensities measured at the particular pixel. Itis understood that any calculation to remove noise and to isolate thestable intensity value for the pixel can be employed without departingfrom the present teachings such as selecting the median intensity valueor ignoring outliers and then averaging, or estimating a temporal driftin illumination level and correcting noise values to offset temporaldrift. It is understood that any means of determining the stableintensity can be employed. Similarly, the statistical dispersions forlike intensities is calculated as a measure of noise, for example bycalculating the standard deviation of the measured intensities in thesample photometric intensity readings.

FIG. 4 is a graph of radiant energy as a function of position along aone dimensional array of photometric sensor pixels. A linear array ofpixels 414 is illustrated in FIG. 4 to show how a non-uniformillumination field can be provided to produce varying levels ofintensity at the discrete pixels in a photometric detector. Generally,pixels in a photometric detector have a detector saturation level atwhich the pixel cannot store intensity values that are above thedetector saturation level. Illumination field distribution curve 408 isshown ranging from dark (at segment 402) to saturated at segment 403.

FIG. 5 illustrates an exemplary photon transfer curve. The read noisesegment 505 of the photon transfer curve is the portion of the photontransfer curve corresponding to noise levels where the photometricdetector is dark or exposed to very low levels of intensity. Such noiseis considered to be associated with the photometric detector itself andthe circuitry used to read values from the photometric detector. Segment504 is called the shot-noise region that illustrates noise produced inthe photometric detector caused by random arrival of electromagneticenergy on the photometric detector. Finally, segment 506 is the fixedpattern noise or pixel non-uniformity region that results fromsensitivity differences between pixels.

Based on the signal to noise relationships established by measuring thephoton transfer curve for a photometric detector, variouscharacteristics of the photometric detector can be calculated. Suchcharacteristics include photometric detector gain, dynamic range,full-well capacity or saturation intensity, and read noise. Thesecharacteristics can be evaluated to determine whether the photometricdetector can provide measurements that allow the instrument thatcontains the photometric detector to operate within the instrument'sstated limits of detection.

In various embodiments, the above exemplary photon transfer curve of aphotometric detector is measured while the photometric detector isinstalled in an instrument. This allows observation of the effect of theinstrument on the detector. In various embodiments, the photon transfercurve of a photometric detector is measured using highly non-uniformillumination fields eliminating the need for costly test fixturesdesigned to produce highly uniform illumination fields. The presentteachings enable highly quantitative verification of an instrument'sphotometric detector without removing the detector from the instrument.In various embodiments, the photon transfer curve of a photometricdetector can be measured with a lens assembly attached to the detector,for example to simplify manufacturing procedures. The photon transfercurve of a photometric detector can be measured with thousands of timeshigher resolution than methods employed by the prior art.

While the present teachings are described in conjunction with variousembodiments, it is not intended that the present teachings be limited tosuch embodiments. On the contrary, the present teachings encompassvarious alternatives, modifications, and equivalents, as will beappreciated by those of skill in the art.

1.-13. (canceled)
 14. A method for assessing the operationalcharacteristics of a photometric sensor, the method comprising:receiving a series of electromagnetic signals of non-uniform intensitycaptured with a photometric sensor having a plurality of pixels whereinat least a portion of each the series of electromagnetic signals isdistributed over two or more pixels; assessing intensity variationsbetween each of the series of electromagnetic signals on apixel-by-pixel basis; approximating a noise level and an illuminationlevel associated with the photometric sensor based on the intensityvariations in the series of electromagnetic signals for each pixel; andassessing the illumination level versus the noise level associated withthe photometric sensor to determine its operating characteristics. 15.The method of claim 1, wherein the noise level for each pixel iscalculated as a standard deviation of the received series ofelectromagnetic signals for that pixel.
 16. The method of claim 1,wherein the illumination level for each pixel is calculated by averagingthe intensities for the received series of electromagnetic signals forthat pixel.
 17. The method of claim 1, wherein the photometric detectoris calibrated on the basis of the assessed illumination levels and noiselevels for each pixel.
 18. The method of claim 1, wherein theoperational limits of detection for the photometric sensor aredetermining on the basis of the photometric sensors operatingcharacteristics.
 19. The method of claim 18, wherein the operatingcharacteristics are selected from the group consisting of: gain, fullwell capacity, read noise, dynamic range, shot-noise.
 20. The method ofclaim 1, wherein each electromagnetic signal is spatially non-uniform inintensity.
 21. The method of claim 20, wherein each electromagneticsignal is temporally constant.
 22. The method of claim 20, wherein theseries of electromagnetic signals comprises an electromagnetic signalthat varies in spatial intensity over time to produce a time varyingillumination field.
 23. An apparatus for assessing the operatingcharacteristics of a photometric sensor, the apparatus comprising: aphotometric sensor having a plurality of pixels that receives a seriesof electromagnetic signals wherein at least a portion of eachelectromagnetic signal is distributed over two or more pixels andwherein each portion of each electromagnetic signal associated with aparticular pixel has a corresponding intensity; and a photometric sensorinterface circuit that assesses intensity variations in the receivedportions of the electromagnetic signals distributed over various pixelson a pixel-by-pixel basis and approximates a noise level and anillumination level based on the intensity variations to determine theoperating characteristics of the photometric sensor.
 24. The apparatusof claim 23, wherein the photometric sensor is selected from the groupconsisting of: a charge coupled device (CCD), a complimentary metaloxide semiconductor (CMOS) image sensor, a spectrometer and a line-scancamera.